JP3264276B2 - Method for detecting nucleic acid fragment molecular weight separation pattern - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for analyzing DNA and RNA, which are genetic materials of humans and other organisms. In particular, the present invention relates to a method for detecting a nucleic acid fragment molecular weight separation pattern, which is effective for determining the nucleotide sequence of DNA or RNA or measuring the polymorphism of a restriction enzyme fragment based on the diversity of individual nucleotide sequences.

[0002]

2. Description of the Related Art Methods for automatically determining the base sequence of DNA, that is, sequencing using fluorescence detection, are described in Biotechnics, Vol. 5 (1987), pp. 342 to 348 (Bio-Techniques 5 (1987) 342). ~ 34
8). In this method, a fluorescent substance is labeled on a primer used for a DNA sequencing reaction.
At this time, the primers used for the four kinds of reactions corresponding to the four kinds of bases of the DNA are labeled with different fluorophores, respectively, and the generated DNA fragments are separated by electrophoresis on the same migration path. . When the gel, which is the electrophoresis medium, is electrophoresed for a certain distance, a DNA fragment labeled with a fluorescent substance is excited by a laser beam. Fluorescent substances are identified by detecting the fluorescence emitted at this time and selecting the wavelength.
Obtain the molecular weight separation pattern of the NA fragment. The base sequence of the DNA is determined by analyzing this pattern.

A method of measuring the molecular weight distribution of a restriction enzyme digested fragment of DNA by fluorescence detection is known as genomics 4.
Volume (1989), pp. 129-136 (GENOMICS
4 (1989) 129-136). In this method, a cosmid or a plasmid DNA is cleaved with a restriction enzyme, and a fluorescent-labeled double-stranded DNA oligomer is bound to the cleavage sites at both ends of the fragment by a ligation reaction, and used in the method described in the preceding paragraph. Using the same apparatus as described above, the molecular weight separation pattern of the generated fluorescent substance-labeled DNA fragment was obtained.

[0004]

SUMMARY OF THE INVENTION In electrophoresis, DN
In the above-described conventional technique in which the nucleotide sequence of A is determined or the molecular weight distribution of the restriction enzyme fragment is measured using fluorescence detection, the wavelength of excitation light cannot be sufficiently separated from the wavelength at which fluorescence is detected. There has been a problem that the background fluorescence from the component or the gel is insufficiently reduced, thereby determining the detection sensitivity. Further, since four kinds of phosphors are excited by a single laser beam, there is a problem that the detection sensitivity is determined by the phosphor having the lowest excitation efficiency among the four kinds. Conventionally, it has been substantially impossible to excite four or more types of phosphors with a single laser beam.

SUMMARY OF THE INVENTION An object of the present invention is to provide an electrophoretic separation and detection method for measuring a molecular weight separation pattern of DNA by fluorescence detection in electrophoresis with high sensitivity. To excite the DNA
It is to provide a method for identifying and detecting a sample. It is another object of the present invention to provide a method for detecting a molecular weight separation pattern of nucleic acid fragments, which performs highly sensitive measurement of a DNA molecular weight separation pattern by multicolor detection of fluorescence.

[0006]

According to the present invention, in order to achieve the above object, two or more kinds of fluorescent substances are labeled on a nucleic acid fragment sample for which a molecular weight separation pattern is measured by electrophoresis, and at least one of the fluorescent substances is labeled. By measuring the molecular weight separation pattern by detecting the fluorescence emitted from another type of fluorescent substance by transferring the energy of the fluorescent light generated by exciting the fluorescent substance. In addition, two or more types of nucleic acid fragment samples are separated by molecular weight at a place where they have been electrophoresed at a fixed distance in an electrophoresis medium. Identification of a nucleic acid fragment sample is performed based on a difference in emission wavelength of another type of labeled fluorescent material that emits light due to transfer of fluorescence energy generated by exciting at least one of the labeled fluorescent materials. .

In order to achieve the above object, as another method, two or more types of fluorescent substances are labeled on a plurality of types of nucleic acid fragment samples to be identified and detected, and the labeled fluorescent materials are labeled for each type of the nucleic acid samples. The distance between them is changed, at least one of the phosphors is excited, and the intensity ratio of fluorescence from the phosphor and another phosphor that emits light by energy transfer is measured.

In particular, for DNA sequencing, a primer, which is a single-stranded DNA oligomer used in a sequencing reaction, is labeled with two kinds of fluorophores, and in order to identify four kinds of bases, a labeled fluorescent primer is used. The distance between the bodies is set to have four types of distances of 15 bases or less.

For the efficiency of energy transfer between two kinds of phosphors, which are an energy donor and an energy acceptor, a formula by Von Th. Foerster is known (Anneren der Fusig series).
6.2 (1948) pp. 55-75 (Annalen der
Physik. Folge. Band 2. (1948) pp. 55-7
5)). According to this, the energy transfer rate is inversely proportional to the sixth power of the distance between the energy donor and the acceptor, and is proportional to the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. It is also affected by the orientation between donor and acceptor.

An experimental study of the distance dependence between energy transfer donors and acceptors has been reported by L. Stryer and RP Haugland (Proceedings of the National Academy of Sciences of Science). USA 58 (196
7 years) pp. 719 to 726 (Proc. Natl. Acad. Sc)
i. USA 58 (1967) 719-726)). This is the result of changing the distance between the donor and acceptor at both ends by changing the number of peptides in the polypeptide oligomer. The distance dependence of the energy transfer agrees well with Forster's equation. It is shown that. The energy transfer efficiency is 100% when the distance between the donor and the acceptor is 1.2 nm, and 16% when the distance is 4.6 nm.
Has been obtained.

Therefore, single-stranded or double-stranded DNA
If the distance between the energy donor and the acceptor phosphor is several nm or less and the overlap between the emission spectrum of the former and the absorption spectrum of the latter is large, energy transfer will occur between the two. Moreover, 10% or more 1
Energy transfer with an efficiency close to 00% can be expected. That is, when the phosphor serving as the energy donor is excited, highly efficient light emission from the energy acceptor can be observed. Also,
Compared to the case where only the fluorescent substance serving as the energy donor is labeled, the emission wavelength shifts to the longer wavelength side, so that the wavelength of the excitation light is separated from the wavelength of the emission light. Therefore, the measurement is less susceptible to scattering of excitation light. For the above reasons, highly sensitive fluorescence measurement can be realized.

Further, when selecting a plurality of luminous bodies which can be excited at the same wavelength, if light emission by energy transfer is used, a plurality of acceptors receiving energy transfer with high efficiency to the same energy donor can be selected. Combination, or combination of multiple phosphors that can be excited at the same wavelength with high efficiency and receptors that receive energy transfer from each with high efficiency, etc., are possible, and high efficiency excitation is possible.
That is, the range of choice of the fluorescent substance that can be detected with high sensitivity is expanded.

The case of energy transfer between two molecules between an energy donor and an acceptor has been described above. However, the shift between the excitation wavelength and the emission wavelength can be increased, and the number of illuminants that can be detected simultaneously can be increased. To do this, energy transfer between three or more molecules can be used. That is, first, the fluorescent material on the shortest wavelength side is excited, and energy is transferred between the fluorescent material and the second fluorescent material having an excitation spectrum that overlaps greatly to excite the fluorescent material. Next, energy transfer occurs between the fluorescence spectrum of the second phosphor and the third phosphor having an excitation spectrum having a large overlap, thereby exciting the second phosphor. By sequentially exciting the phosphors having the fluorescence spectrum on the long wavelength side in this manner, it becomes possible to increase the shift between the excitation wavelength and the emission wavelength, and to increase the number of simultaneously detectable phosphors.

On the other hand, if the distance between the energy donor and the acceptor is changed, the efficiency of energy transfer will also change. That is, by changing the distance between the energy donor and the acceptor, the intensity ratio between the light emission from the energy donor and the light emission from the acceptor can be changed.
Therefore, by measuring the intensity ratio of the luminescence from the energy donor to the luminescence from the acceptor by changing the distance for each type of nucleic acid sample with the same combination of energy donor and acceptor, The sample can be identified.

If the efficiency of energy transfer is about 10% or more, sufficient light emission can be obtained as compared with the case where four kinds of phosphors are excited by a single laser. Therefore, the distance between the energy donor and the acceptor may be about 15 bases or less for both single-stranded DNA and double-stranded DNA. That is, in the double-stranded state, the base pair spacing is 1
It is several nm for 5 bases, and the average distance becomes several nm or less even in a single-stranded state in consideration of chain folding.

In the case of DNA sequencing, by changing the distance between the energy donor and the acceptor for each type of single-stranded DNA oligomer, four types of primers used for the sequencing reaction, ie, for each type of single-stranded DNA oligomer, The base species of DNA fragments of various lengths, which are the products of the sequence reaction, can be identified.

[0017]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First Embodiment A first embodiment according to the present invention will be described below with reference to FIGS. In this example, using λ phage DNA as a model substance, samples obtained by cleaving the λ phage DNA with different combinations of restriction enzymes were labeled with different combinations of fluorophores, and then electrophoresed simultaneously on the same migration path. Differences in patterns are identified by differences in emission wavelength.

In this embodiment, double-stranded DNA oligomers 1a and 1b labeled with two kinds of fluorescent substances which generate energy transfer between them are introduced into a cleavage site of a restriction enzyme HindIII by a ligation reaction. As shown in FIG. 1, fluorescein isothiocyanate (FITC) 2 which can be efficiently excited by an argon ion laser (emission wavelength: 488 nm) is used as an energy donor. As an energy acceptor, tetramethylrhodamine isothiocyanate (TRITC) 3, which has a large overlap with the fluorescence spectrum of FITC2, and sulfolodamine 10
1 sulfonic acid derivative (Texas Red)
4 is used.

The DNA double-stranded oligomer used for the introduction of the labeled fluorescent substance by the ligation reaction is as follows:
The single-stranded portion at the right end matches the cleavage shape of HindIII.
Selectively binds to the cleavage site of the III cleavage fragment.

5'-CGTTTGTAAAACGACGG
CCAGT-3 '3'-GCAACATTTTGCTGCCGGTCAT
The labeling position of the CGA-5 'phosphor is FITC2 at the phosphorus moiety between the 12th C and 13th T from the 3' side of the lower chain.
RITC3 and Texas Red 4 are the portions of the phosphorus between the third and fourth T from 5 'of the upper strand. The method disclosed in JP-A-61-44353 is used for labeling the energy donor and the acceptor with the oligonucleotide.
In this method, the phosphoric acid bond at the fluorescent labeling site of the polynucleotide is replaced by a phosphonic acid bond having a functional group, and the fluorescent group is realized by binding the functional group to the fluorescent substance. This is a technique that allows the introduction of a label to the target.

In this embodiment, the case where the fluorescent label is bonded to the phosphorus atom of the nucleotide skeleton has been described. However, the label position is not limited to this.
8 (1987) pages 336-341 (Science, 2
38 (1987) pp. 336 to 341), and it is also possible to label the base using a method similar to that described in the above.

The double-stranded oligomer is prepared by separately synthesizing each of the constituent chains, and thereafter annealing to form a double-stranded chain.

Fluorescently labeled double-stranded DNA oligomer 1
FIG. 2 shows the respective fluorescence spectra of a and 1b. This is a spectrum when excited at 488 nm, which is the oscillation wavelength of an argon laser, and a sufficient amount of energy transfer occurs between each energy donor and acceptor. Moreover, an emission intensity equal to or higher than that obtained when excited by a He-Ne laser having an oscillation wavelength of 543 nm that can excite TRITC3 and Texas Red 4 in common can be obtained. Therefore, each laser (argon laser, 〜１０10 mW,
Considering the intensity of the excitation light obtained with a He-Ne laser (up to 1 mW) and the intensity of the scattered light due to the difference between the excitation wavelength and the emission wavelength, more sensitive fluorescence detection is realized using energy transfer. You can do it.

The preparation of a DNA sample is performed in the following procedure. λ
To a degradation product (λ / HindIII; commercially available product) 5 obtained by digesting the phage DNA with the restriction enzyme HindIII, about ten times the amount of each of the fluorescently labeled double-stranded DNA oligomers 1a and 1b was added.
Ligation reaction 6 is performed at 16 ° C. for 30 minutes using a commercially available ligation kit, and the reaction product is purified by ethanol precipitation. Next, the reaction product containing Texas Red 4 was added to 50 mM Tris-HCl (pH 7.5), 1
0 mM MgCl ₂ , 1 mM Dithiothreitol, 50 mM
Restriction enzyme in 37 ° C buffer consisting of NaCl
A restriction enzyme digestion reaction 7 is performed with HinfI, and the reaction product is purified by ethanol precipitation.

Next, these reaction products are mixed 8, injected into the same electrophoresis path, and subjected to electrophoresis 9. Glass plate 6
A 3% acrylamide gel is used as the gel 60 for electrophoresis sandwiched between 1. A voltage of about 30 V / cm is applied between the upper end and the lower end of the gel 60. The measurement of the fluorescence is performed by causing an argon laser beam 63 from a laser light source 62 to be incident perpendicularly to the migration direction of the DNA fragment 70 from the end surface of the gel 60 at a migration distance of 10 cm by a reflecting mirror 64. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is received by the fluorescent two-color detection device. The fluorescent two-color detection device includes a prism 65 that divides a linear image of a laser beam irradiation site into two, an optical filter 66 through which a beam from each image passes, and a lens system that forms an image. 6
7 and a two-dimensional detector 68. The optical filter 66 has a center wavelength of 580 nm for transmitting light emitted from TRITC3 mainly and a half value width of 40 nm, and a center wavelength 615 for transmitting light emitted mainly from Texas Red 4 mainly.
The half-width of 30 nm was used. Two-dimensional detector 6
8 is a CCD with an image intensifier
A camera was used. The signal from the two-dimensional detector 68 is processed by the data processing device 69, and the light emission from each phosphor is identified by color correction calculation. In the color correction calculation, the emission intensity of each phosphor is obtained by a linear combination of the emission intensity values of the two types of emission spectra observed, based on the parameters obtained from the respective emission spectra and the transmission characteristics of the optical filter. FIG. 3 shows an example of the observed signal. The signal from the fragment obtained by digesting the λ phage DNA with HindIII and the signal from the fragment further digested with Hinfl can be clearly identified and detected.

In this embodiment, λ phage DNA is used as a model sample, but this technique is effective for the following uses. First of all, a certain restriction enzyme digest of DNA whose molecular weight distribution of the restriction enzyme fragment is to be investigated and a specific restriction enzyme digest of which the molecular weight distribution of the fragment is known are separated into different fluorescent substances. Labeling is performed in combination, and comparison of each electrophoresis pattern is performed by identifying each emission wavelength. This makes it possible to determine the molecular weight distribution of the DNA fragment. As another example, for example, genomic DNA of bacteria of different strains
Are labeled with different combinations of phosphors,
The comparison of the electrophoretic patterns of the digested fragments by the same combination of the same restriction enzymes was performed, and the difference in the wavelength of the DNA digested fragment due to the difference in the strain was determined by identifying the emission wavelength of each fluorophore and determining the difference. Can be selectively detected. The latter example is also effective when comparing individuals with a genetic disease and normal individuals.

Second Embodiment A second embodiment according to the present invention will be described with reference to FIGS. The present embodiment is directed to the determination of the base sequence of DNA. The DNA sequencing reaction is based on the Sanger method. A combination of the following four kinds of fluorescent substances is used as primers 33, 34, 35, 36 for sequencing corresponding to four kinds of bases of DNA. The first two are the same combination as in the first embodiment, FITC28 and TRITC2.
9, and Texas Red 30, and the remaining two were a combination of a succinylfluorescein derivative (SF526) 31 having an emission peak at FITC 28 and 526 nm, and a phthalocyanine derivative 32 having an emission peak at FITC 28 and 680 nm. . FITC
28 and each of the above phosphors are labeled on the primer with a distance of about 4 bases. The labeling of the oligonucleotide with the energy donor and the acceptor was performed in the same manner as in the first embodiment.
The method disclosed in JP-A-61-44353 is used. The following labeling method is used to label the energy donor and acceptor one by one on the oligonucleotide. First, the oligonucleotide having a phosphonic acid introduced at the above-mentioned label position is labeled with F
A reaction for labeling ITC28 is performed. At this time, the reaction product was monitored at any time by liquid chromatography, and the absorbance at 260 nm derived from DNA and 490 nm derived from FITC28 were monitored.
By comparing the absorbances of the two, the reaction is stopped at a stage where FITC 28 has been introduced to some extent and two FITC 28 have not been introduced so much. The reaction product is subjected to electrophoresis in an acrylamide gel to separate an unlabeled oligonucleotide and one into which one and two FITC28 have been introduced, cut out a gel containing a part into which one FITC28 has been introduced, and contained the gel therein. The oligonucleotide to be purified is purified. The FITC-labeled DNA oligomer thus produced is subjected to a reaction for labeling TRITC 29, Texas Red 30, SF526 31, and phthalocyanine derivative 32, respectively. By the above reaction process, TRITC29, Texas Red 3
Thus, DNA oligomers 33, 34, 35 and 36 in which one of 0, SF526 31, and the phthalocyanine derivative 32 and one FITC 28 are labeled are obtained. The saxinylfluorescein derivative and the phthalocyanine derivative are disclosed in JP-A-1-180455 and PCT application WO8, respectively.
It can be synthesized by the method disclosed in JP-A-8 / 04777.

The sequence reaction 37 is performed according to a usual reaction protocol. Reaction products 38, 39,
As shown in FIG. 5, electrophoresis 9 is performed on the same electrophoresis path of acrylamide gel 60 under denaturing conditions in which urea is mixed with urea, as shown in FIG.
The fragment A is separated according to its molecular weight, and a laser beam 47 is incident from the side of the gel 60 perpendicular to the migration path, and the DNA
The fluorescence emitted from the fragments is detected. The measurement 44 of the light emission from the labeled fluorescent material on the DNA fragment is detected by the same combination of the excitation laser light, the electrophoresis apparatus, and the photodetector as in the first embodiment. The difference from the first embodiment lies in the image division portion of the emission image shown in FIG. 5. In this embodiment, in order to detect four types of light emission, a pentagonal prism 48 and four types having different transmission characteristics are used. The image 52 is formed on the image plane 51 by the image forming lens system 50 using the optical filter 49 described above.

In this embodiment, as an alternative to the fourth probe, as shown in FIG. 6, three kinds of fluorescent materials, FITC28, Texas Red 30, and phthalocyanine derivative 32, are separated by about 4 bases each to label the primer. , T reaction primer 36, and light emission from the phthalocyanine derivative 32 can be observed with high efficiency.

In this example, energy transfer between three molecules is used. That is, first, the fluorescent substance FITC28 having the emission peak on the shortest wavelength side is excited, and energy transfer is performed between the second fluorescent substance, Texas Red 30, and the second fluorescent substance having an excitation spectrum overlapping the fluorescent spectrum of this fluorescent substance. Wake up and excite this. Next, a phthalocyanine derivative 32, a third phosphor having an excitation spectrum that overlaps with the fluorescence spectrum of the second phosphor.
Energy transfer occurs between and excites it.
By sequentially exciting the phosphors having the fluorescence spectrum on the long wavelength side in this manner, the shift between the excitation wavelength and the emission wavelength is increased.

Third Embodiment Hereinafter, a third embodiment according to the present invention will be described with reference to FIG. The present embodiment is directed to the determination of the base sequence of DNA. The DNA sequencing reaction is based on the Sanger method. In this embodiment, the following primers are used as sequencing primers. As a combination of phosphors, an argon ion laser (with an oscillation wavelength of 4) was used as an energy donor.
Fluorescein isothiocyanate (FITC) 71, which can be efficiently excited at 88 nm), as an energy acceptor, the excitation spectrum largely overlaps with the fluorescence spectrum of FITC 71, and the maximum of the fluorescence spectrum is FITC 71.
And a sulfonated chloride derivative (trade name: Texas Red) 72 of sulfolodamine 101 which is separated from that of the above by nearly 100 nm. Four types of primers 73, 74, 7
FIT on primers corresponding to 5,76
Change the distance between C71 and Texas Red 72. That is, FITC71 and Texas Red 72 are labeled with a distance of 2, 5, 9, or 12 bases apart. The nucleotide sequence of the primer was 30 mer immediately upstream of the cutting site of the M13 phage with the restriction enzyme.

As in the first embodiment, the labeling of the oligonucleotide with the energy donor and the acceptor was performed in the same manner as in the first embodiment.
The method disclosed in JP-A-61-44353 is used.
The following labeling method is used to label the energy donor and acceptor one by one on the oligonucleotide. First, a reaction of labeling FITC71 with the oligonucleotide having a phosphonic acid introduced at the label position is performed. At this time, the reaction product is monitored at any time by liquid chromatography, and DN
Absorbance at 260 nm from A and 49 from FITC71
By comparing the absorbances at 0 nm, the reaction is stopped at a stage where FITC71 has been introduced to some extent and two FITC71s have not been introduced much. The reaction product is subjected to electrophoresis in an acrylamide gel to separate an unlabeled oligonucleotide from one into which one and two FITC71 have been introduced, cut out a gel containing a portion into which one FITC71 has been introduced, and contained the gel therein. The oligonucleotide to be purified is purified. A reaction for labeling Texas Red 72 with the FITC-labeled DNA oligomer thus generated is performed. Through the above reaction process, DNA oligomers 73, 74, 75 and 76 in which one FITC 71 and one Texas Red 72 are labeled are obtained.

The DNA oligomer prepared as described above,
That is, a sequence reaction 37 is performed for each base type using the primers 73, 74, 75, and 76. Next, these reaction products 38, 39, 40 and 41 are mixed 8 and subjected to electrophoresis 9 in the same migration path. As the gel 60 for electrophoresis, a 4.5% acrylamide gel sandwiched between glass plates 61 and mixed with urea as a denaturant is used. A voltage of about 30 V / cm is applied between the upper end and the lower end of the gel 60. Fluorescence was measured at gel distance of 30 cm at a migration distance of 30 cm.
Argon laser light 6 from the laser light source 62 from the end face
3 is performed by using a reflecting mirror 64 and making the light incident perpendicularly to the migration direction. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is received by the fluorescent two-color detection device. The fluorescent two-color detection device is the same as that shown in FIG. 1 described in the first embodiment. The optical filter 66 has a central wavelength of 520 nm for transmitting light emitted from the FITC 71 and a half-value width of 30 nm, and a central wavelength 61 for transmitting light emitted from Texas Red 72.
5 nm and a half value width of 30 nm were used.

When these DNA fragments of each base type are excited by an argon laser having an oscillation wavelength of 488 nm, FITC7
FI with a peak at 520 nm due to the difference in energy transfer efficiency corresponding to the distance between 1 and Texas Red 72
The four types of primers 38, 39, 40, and 41 can be identified by measuring the intensity ratio of the emission of TC71 and the emission of Texas Red 72 having a peak at 620 nm. That is, the signal from the two-dimensional detector 68 is processed by the data processing device 69, the light emission from each phosphor is identified, the intensity ratio is calculated, and the base type is specified.

In this embodiment, the sequencing by electrophoresis using the flat gel 60 has been described, but the present invention is not limited to this. In particular, from 50 μm to 1
In electrophoresis in a gel or electrolyte in a single capillary tube of about 00 μm, ie, sequencing by capillary electrophoresis, it is essential to simultaneously migrate four types of samples for each base type, so that the effect is large.

Further, in this embodiment, FITC 71 and Texas Red 72 which can be excited by an argon ion laser as a combination of phosphors which cause energy transfer therebetween.
However, the present invention is not limited to this. In particular, by selecting an appropriate combination of phthalocyanine derivatives capable of changing its fluorescence spectrum in the range of 650 nm to 750 nm by changing the functional group or the central metal, it becomes possible to excite with a semiconductor laser. In addition, downsizing of the apparatus and reduction in price can be realized.

Fourth Embodiment A fourth embodiment according to the present invention will be described with reference to FIG. In this embodiment, a simultaneous sequence of the sense strand and the antisense strand of DNA in the same migration path is realized. In this example, DNA inserted into plasmid PUC18 was used.
Target the sample. Primer for sense strand 87,8
8, 89 and 90 are succinylfluorescein derivatives (SF526) 83 which can be efficiently excited by an argon ion laser as an energy donor and have an emission peak at 526 nm whose fluorescence spectrum largely overlaps with the excitation spectrum of Texas Red 84. , Labeled with Texas Red 84 as an energy acceptor. The distances between the energy donor and the acceptor in the above four primers are 2, 5, 9, and 12 as in the third embodiment. A primer for sequencing in the opposite direction, a primer 91 for antisense strand direction,
92, 93, and 94 can also be efficiently excited by an argon ion laser as energy donors, and
26 can be clearly distinguished from 83 fluorescence spectra, 50
A succinylfluorescein derivative (SF505) 85 having an emission peak at 5 nm, as an energy acceptor, the excitation spectrum of which overlaps greatly with the fluorescence spectrum of SF505 85 and the maximum of the fluorescence spectrum is 580 nm, and that of SF505 85, SF526 83 and Texas Red 84 at 580 nm. Labeled with clearly distinguishable tetramethylrhodamine isothiocyanate (TRITC) 86. The distance between the energy donor and the acceptor in the above four primers was the same as that used in the third embodiment.
They are 2, 5, 9 and 12 bases. The base sequence is 30 bases immediately downstream of the cutting site of PUC. Note that SF505 and SF526 are disclosed in
It can be synthesized by the method disclosed in -180455.

The sequence reactions 95 and 6 are carried out according to a usual reaction protocol. Reaction product 97a for each base species,
97b is mixed 8 and, as shown in FIG.
In the same electrophoresis path in the acrylamide gel 60 under denaturing conditions mixed with urea,
DNA fragments are separated by their molecular weight. The measurement 44 of the emission of the fluorescent substance based on the energy transfer from the fluorescent label on the DNA fragment is performed by the combination of the laser beam for excitation, the electrophoresis apparatus, and the photodetector similar to the first embodiment shown in FIG. To detect. The difference from the first embodiment is the image division portion of the light emission image shown in FIG. 5. In this embodiment, a pentagonal prism 48 and a pentagonal prism 48 are used to detect four types of light emission excited by the laser beam 47. Four types of optical filters 49 having different characteristics are used. With the above optical system, it is possible to identify and detect light emission from four types of phosphors. SF526 83 and Texas Red 8 at the data processor 69
By calculating the emission intensity ratio between 4, SF50585 and TRITC26, the direction of the DNA to be analyzed and the sequence of the base type can be specified.

Fifth Embodiment A fifth embodiment according to the present invention will be described with reference to FIG. In this example, using λ phage DNA as a model substance, samples obtained by cleaving the λ phage DNA with different combinations of restriction enzymes were labeled with energy donors and acceptors whose distances were different from each other, and were labeled on the same migration path. Simultaneous electrophoresis is performed, and the difference in the pattern is identified based on the difference in the emission intensity ratio between the donor and the acceptor.

In the present embodiment, double-stranded DNA oligomers 1c and 1d labeled with two kinds of fluorescent substances that generate energy transfer between them are introduced into the cleavage site of the restriction enzyme HindIII by the ligation reaction 6. FITC is used as an energy donor, and Texas Red 4a, 4b is used as an energy acceptor. The DNA double-stranded oligomer used to introduce the labeled fluorescent substance by the ligation reaction 6 has the structure of the following SEQ ID NO: 1, and the right-hand single-stranded portion is HindII.
It is consistent with the cleavage shape of I, and selectively binds to the cleavage site of the HindIII cleavage fragment by ligation reaction 6.

5'-CGTTTGTAAAACGACGG
CCAGT-3 '3'-GCAACATTTTGCTGCCGGTCAT
Regarding the labeling position of the CGA-5 'fluorescent substance, FITC2 is the phosphorus portion between the third T and the fourth T from the 5' side of the upper strand, and Texas Red 4a and 4b are the lower strands, respectively. Phosphorus portion between 7th T and 8th T from 3 'side, 3'
A portion of phosphorus between the twelfth C and the thirteenth T from the side, and the same method as in the third embodiment is used for labeling the oligonucleotide of the energy donor and the acceptor.
For the double-stranded oligomer, each of the constituent chains is separately synthesized, and later converted into a double-stranded chain by annealing.

The preparation of a DNA sample is performed according to the following procedure. λ
To a degradation product (λ / HindIII; commercially available product) 5 obtained by digesting the phage DNA with the restriction enzyme HindIII, about 10 times the amount of each of the fluorescently labeled double-stranded DNA oligomers 1c and 1d was added.
Ligation reaction 6 is performed at 16 ° C. for 30 minutes using a commercially available ligation kit, and the reaction product is purified by ethanol precipitation. Next, the reaction product containing Texas Red 4b between the twelfth C and the thirteenth T from the 3 'side was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl ₂ , 1 mM Dithiothreitol, 50 mM N
Restriction enzyme Hinf in a buffer of 37 ° C consisting of aCl
A restriction enzyme digestion reaction 7 is carried out by I, and the reaction product is purified by ethanol precipitation.

Next, these reaction products are mixed 8 and subjected to electrophoresis 9 in the same electrophoresis path under the following conditions. As the gel for electrophoresis 9, a 3% acrylamide gel is used. Apply a voltage of about 30 V / cm between the top and bottom of the gel,
The measurement of light emission 44 is performed by injecting argon laser light perpendicularly to the migration direction from the end face of the gel at a migration distance of 10 cm. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is detected by a fluorescent two-color detection device as shown in FIG. The same two-color fluorescence detector as that of the third embodiment is used. By analyzing data of the emission intensity around 520 nm and the emission intensity around 620 nm, the signal from the fragment obtained by digesting the λ phage DNA with HindIII and the signal from the fragment obtained by digesting the λ phage DNA with HinfI are clearly distinguished and detected. be able to.

In this example, λ phage DNA was used as a model sample, but this technique is effective for the following uses. First, a certain restriction enzyme digest of DNA whose molecular weight distribution of the restriction enzyme fragment is to be investigated and a specific restriction enzyme digest of which the molecular weight distribution of the restriction fragment is known are separated from each other by distance. Labeling is performed with a combination of different phosphors, and the comparison of each electrophoresis pattern is performed by identifying the emission intensity of each phosphor. Thereby, the molecular weight distribution of the DNA fragment can be obtained. As another example, for example, genomic DNAs of different bacterial strains are labeled with a combination of fluorophores having different distances from each other, and a comparison of electrophoresis patterns of digested fragments with the same combination of restriction enzymes is performed.
By discriminating the respective luminescence intensities and determining the difference, it is possible to selectively detect only the difference in the DNA digestion fragment due to the difference in the strain. The latter example is also effective when comparing individuals with a genetic disease and normal individuals.

[0045]

According to the present invention, it is possible to detect fluorescence in the same migration path in electrophoresis by sufficiently separating the wavelength of the excitation light and the wavelength of the emission light. Can be realized. Further, since various kinds of phosphors can be excited with a single laser beam with high efficiency, it is possible to measure a high-sensitivity and high-throughput measurement of a DNA molecular weight separation pattern.

Further, according to the present invention, discrimination of four or more DNA samples can be realized by two kinds of phosphors (dyes), and a large number of DNA samples can be simultaneously discriminated by a single laser beam in the same migration path. And can be detected. In addition, more sensitive detection can be realized than in the case where the detection is performed by changing the fluorescent substance for each type of DNA sample. Therefore, it is possible to greatly improve the throughput of DNA sequencing. For example, simultaneous sequencing of four or more kinds of DNA samples, simultaneous sequencing of sense strand and antisense strand, and the like can be performed. Further, simultaneous measurement of the molecular weight distribution of restriction enzyme cleavage fragments of four or more DNA samples can be realized. In particular, in the case of sequencing using capillary electrophoresis, the effect of the present invention is great because it is necessary to identify four types of samples for each base type.

[0047]

[Sequence List] SEQUENCE LISTING <110> HITACH, LTD. <120> Method for Detecting Molecular Weight Separation Pattern of DNA Fragments <130> H91010623A <160> 1 <210> 1 <211> 21 <212> DNA <213> artificial Sequence <220><222> 21 <223> Complementary strand has a single stranded-DNA "3'-tcga -5 '" extending from this point in a direction 3' → 5 ', and double stranded-DNA oligomer can be ligated with a double stranded-DNA fragment digested by HindIII. <400> 1 cgttgtaaaa cgacggccagt 21
Sequence Listing Free Text (1) Description of other related information regarding the sequence of SEQ ID NO: 1 has a single-stranded "3'-tcga-5 '"
A double-stranded DNA that can bind to a cleavage site of indIII by a ligation reaction.

[Brief description of the drawings]

FIG. 1 is a schematic view showing a first embodiment according to the present invention.

FIG. 2 is a graph showing characteristics of components in the first embodiment according to the present invention.

FIG. 3 is a graph showing an example of a result in the first example according to the present invention.

FIG. 4 is a schematic diagram showing a second embodiment according to the present invention.

FIG. 5 is a configuration diagram of a detection unit according to a second embodiment of the present invention.

FIG. 6 shows an alternative example of the primer for T reaction in the second embodiment according to the present invention.

FIG. 7 is a schematic diagram showing a third embodiment according to the present invention.

FIG. 8 is a schematic diagram showing a fourth embodiment according to the present invention.

FIG. 9 is a schematic view showing a fifth embodiment according to the present invention.

[Explanation of symbols]

1a, 1b, 1c, 1d: fluorescently labeled double-stranded oligomer, 2, 28, 71: FITC, 3, 29, 86 ... T
RITC, 4, 4a, 4b, 30, 72, 84 Texas red, 5 λ / HindIII, 6 ligation reaction, 7 restriction enzyme digestion reaction, 8 mixed, 9 electrophoresis, 19 receptor TRITC Spectrum at the time of, 20
... spectrum when the receptor is Texas Red, 21 ... F
ITC peak, 22 ... TRITC peak, 23 ... Texas red peak, 24 ... 580 nm signal, 25
... signal of 615 nm, 26 ... signal of TRITC, 27 ...
Texas Red signal, 31,83 ... SF526,32
... Phthalocyanine derivative, 33,73 ... A reaction primer, 34,74 ... C reaction primer, 35,75 ...
G reaction primer, 36, 76 ... T reaction primer, 37 ... sequence reaction, 38 ... A reaction product, 39
... C reaction product, 40 ... G reaction product, 41 ... T reaction product, 44 ... Measurement of light emission, 47 ... Laser light, 48 ... Pentagonal prism, 49 ... Optical filter, 50 ... Imaging lens system, 51 ... Imaging surface, 52 image, 60 gel, 61 glass plate, 62 laser light source, 63 laser light, 64 reflecting mirror, 65 prism, 66 optical filter, 67 imaging lens system, 68 optical system 2D detector, 69 data processing device, 70 DNA fragment, 85 SF505, 87, 8
8, 89, 90 ... sense strand direction primer, 91, 9
2, 93, 94 ... antisense strand direction primer, 95
... sense strand direction sequence reaction, 96 ... antisense strand direction sequence reaction, 97a, 97b ... sequence reaction product.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 15/00 A (72) Inventor Kazuko Kawamoto 1-280 Higashi Koikebo, Kokubunji-shi, Tokyo Hitachi, Ltd. Central Research Laboratory, Hitachi, Ltd. (72) Inventor Satoshi Takahashi 1-280 Higashi-Koigakubo, Kokubunji-shi, Tokyo Hitachi, Ltd., Central Research Laboratory (72) Inventor Tetsuo Nishikawa 1-280 Higashi-Koikekubo, Kokubunji-shi, Tokyo Hitachi, Ltd. Central Research Laboratory, Hitachi, Ltd. ⁷ , DB name) C12Q 1/68 ZNA G01N 21/64 G01N 27/447 G01N 33/50 C12N 15/09 JICST file (JOIS)

Claims (8)

(57) [Claims] (1) a step of obtaining a double-stranded nucleic acid fragment by digesting a double-stranded nucleic acid sample with a first restriction enzyme; (2) separating the double-stranded nucleic acid fragment into a first fraction and a second fraction; And (3) converting the double-stranded DNA oligomer having the first energy acceptor and the first energy donor bound thereto into the double-stranded nucleic acid fragment of the first fraction. Introducing the first double-stranded nucleic acid fragment to obtain a second double-stranded nucleic acid fragment; and (4) combining the second energy acceptor different from the first energy acceptor with the first energy donor. Introducing a single-stranded DNA oligomer into the double-stranded nucleic acid fragment of the second fraction to obtain a second double-stranded nucleic acid fragment; (5) converting the second double-stranded nucleic acid fragment to the second fraction. Digesting with a second restriction enzyme different from the one restriction enzyme to obtain a third double-stranded nucleic acid fragment; (6) Mixing the first double-stranded nucleic acid fragment and the third double-stranded nucleic acid fragment; (7) a mixture of the first double-stranded nucleic acid fragment and the third double-stranded nucleic acid fragment Is separated by electrophoresis, and the separated fragments are irradiated with a laser to detect the fluorescence emitted from the first and second energy acceptors due to the transfer of energy from the first energy donor excited. And detecting the first double-stranded nucleic acid fragment and the third double-stranded nucleic acid fragment. 2. The method for detecting a molecular weight separation pattern of nucleic acid fragments according to claim 1, wherein the first energy donor is attached to a first strand of the double-stranded DNA oligomer.
Is located on the second strand of the double-stranded DNA oligomer, and the second energy receptor is located on the double-stranded DNA
A method for detecting a molecular weight separation pattern of nucleic acid fragments, wherein the nucleic acid fragment is bound to the second strand of the NA oligomer. (3) a step of obtaining a double-stranded nucleic acid fragment by digesting a double-stranded nucleic acid sample with a first restriction enzyme; and (2) separating the double-stranded nucleic acid fragment into a first fraction and a second fraction. (3) dividing the double-stranded DNA oligomer, in which an energy acceptor and an energy donor are bound at a first distance, into the double-stranded nucleic acid fragment of the first fraction To obtain a first double-stranded nucleic acid fragment; and (4) the method wherein the energy acceptor and the energy donor are bound at a second distance different from the first distance. Introducing a single-stranded DNA oligomer into the double-stranded nucleic acid fragment of the second fraction to obtain a second double-stranded nucleic acid fragment; (5) converting the second double-stranded nucleic acid fragment to the second fraction; Digesting with a second restriction enzyme different from the one restriction enzyme to obtain a third double-stranded nucleic acid fragment; Mixing the first double-stranded nucleic acid fragment and the third double-stranded nucleic acid fragment; (7) a mixture of the first double-stranded nucleic acid fragment and the third double-stranded nucleic acid fragment Are separated by electrophoresis, and the separated fragments are irradiated with a laser to detect the fluorescence emitted from the energy donor and the fluorescence emitted from the energy acceptor due to the transfer of energy from the energy donor. The ratio of the intensity of the fluorescence emitted from the energy donor to the intensity of the fluorescence emitted from the energy acceptor, the first double-stranded nucleic acid fragment and the third
A method for detecting a molecular weight separation pattern of a nucleic acid fragment, comprising distinguishing the nucleic acid fragment from a main-stranded nucleic acid fragment for detection. 4. The method for detecting a molecular weight separation pattern of a nucleic acid fragment according to claim 3, wherein the energy donor is the first strand of the double-stranded DNA oligomer, and the energy acceptor is the double strand. A method for detecting a molecular weight separation pattern of nucleic acid fragments, wherein each of the nucleic acid fragments is bound to a second strand of a DNA oligomer. 5. The method of claim 1, wherein the energy donor and the energy acceptor
The energy donor and the
Preparation of multiple nucleic acid fragments linked to energy receptor
Performing the electrophoresis of the plurality of nucleic acid fragments and exciting the nucleic acid fragments.
Transfer of energy from the energy donor
Ri as engineering to detect the fluorescence emitted from the energy receptor
A method for detecting a nucleic acid fragment molecular weight separation pattern, comprising: 6. The method of claim 1 wherein the energy donor and the energy acceptor
The energy donor and the
Preparation of multiple nucleic acid fragments linked to energy receptor
And transferring the plurality of nucleic acid fragments on the same electrophoresis path.
Electrons from the electrophoresed and excited energy donor
Fluorescence from the energy receptor due to energy transfer
Detecting a nucleic acid fragment molecular weight separation pattern. 7. The method of claim 1 wherein the energy donor and the energy acceptor
The energy donor and the
Preparation of multiple nucleic acid fragments linked to energy receptor
Performing the electrophoresis of the plurality of nucleic acid fragments and exciting the nucleic acid fragments.
Transfer of energy from the energy donor
Detecting the fluorescence emitted from the energy acceptor
A method for detecting a nucleic acid fragment molecular weight separation pattern, comprising: 8. The method of claim 1 wherein the energy donor and the energy acceptor
The energy donor and the
Preparation of multiple nucleic acid fragments linked to energy receptor
And transferring the plurality of nucleic acid fragments on the same electrophoresis path.
Electrons from the electrophoresed and excited energy donor
Fluorescence from the energy receptor due to energy transfer
Detecting a nucleic acid fragment molecular weight separation pattern.